Field of the Invention
This invention is in the field of nutriceuticals and nutritional supplements, as well as methods of optimizing the composition of nutriceuticals and nutritional supplements.
Description of the Related Art
Omega-3 Fatty Acids:
Omega-3 fatty acids, also called n-3 poly unsaturated fatty acids (PUFA), have long been suspected of having beneficial effects in humans, particularly with regards to reducing the risk of coronary heart disease, reducing obesity, improving diabetic parameters including blood glucose levels, and improving other parameters of the metabolic syndrome. These fatty acids have a number of beneficial effects, among which is lowering elevated blood triglyceride levels down to more clinically acceptable values (Harris et. al., “Omega-3 fatty acids and coronary heart disease risk: Clinical and mechanistic perspectives” Atherosclerosis. 2008 March; 197(1): 12-24). Omega-3 fatty acids can also assist in weight/fat loss in overweight individuals.
Humans, and indeed essentially all animals, do not synthesize omega-3 fatty acids directly. Instead these fatty acids must be obtained from the diet and therefore are essential dietary nutrients. These fatty acids present in relatively high levels in cold water fish, and other cold water marine animals such as Antarctic krill. The fish themselves do not synthesize the omega-3 fatty acids either, but rather acquire them, usually ultimately from phytoplankton, by virtue of the fish's position on the marine food chain. The commercial omega-3 fatty acids are purified from these marine sources, often by a molecular distillation process to remove unwanted impurities such as mercury, and are sold in both over-the-counter and prescription forms as “fish oil”.
The structures of the various omega-3 fatty acids have been reviewed by Rustan and Devon, “Fatty Acids: Structures and Properties” Encyclopedia of Life Sciences (2001). As they discuss, the most common animal fatty acids can be considered to be carbon chains (typically with a length between about 12 to 22 carbons, and often between 18 and 22 carbons long) with one end terminating at a methyl group, and the other end terminating at a carboxyl group.
The saturated fatty acids are all composed of single carbon-carbon bonds, with the rest of the bonds being primarily occupied with hydrogen, while the unsaturated fatty acids have carbon-carbon double bonds at various positions. The positions of the various carbon atoms in the fatty acid chains are numbered with respect to the terminal methyl group, and from a health standpoint, some of the more important unsaturated fatty acids have double bonds starting between the carbon 3 and carbon 4, and are these called n-3 or omega-3 fatty acids. Other important fatty acids have double bonds starting between the carbon 6 and carbon 7 on the chain, and these are called n-6 or omega-6 fatty acids.
Omega-3 fatty acids with a chain length of 20 atoms are called eicosapentaenoic acid (EPA), while omega-3 fatty acids with a chain length of 22 atoms are called docosahexaenoic acid (DHA). EPA is the precursor for a number of different hormone-like molecules, such as the prostaglandins, and it also has an impact on platelet aggregation. EPA and DHA, when incorporated into cell membranes, are also known to increase membrane fluidity, which may make it easier for blood to circulate, and which may be at least partially responsible for their positive effect on cardiovascular health.
Because it is also cumbersome to repeatedly refer to the EPA and DHA form of the omega-3 fatty acids, in general when the term omega-3 fatty acids are used, the term should also be construed as covering at least the most common EPA and DHA forms of these fatty acids.
In addition to marine sources, certain land plants, such as flax (flax seeds), walnuts, and the like also synthesize omega-3 fatty acids as well, but they often synthesize these omega-3 fatty acids in the form of shorter carbon chains (e.g. 18 carbons), such as alpha-linolenic acid (ALA). Although humans and animals can convert the ALA form to the more useful EPA and DHA form, the process is not particularly fast or efficient, and ALA is generally regarded as being a less favorable omega-3 fatty acid, while EPA and DHA, which do not require chain length conversion, are regarded as being more favorable.
The vast majority of the omega-3 fatty acids naturally occurring in fish oil are primarily present in a triglyceride form in which three fatty acid molecules, one or more of which can be an omega-3 fatty acid, and some of which may be non-omega-3 fatty acids, are bound to a three carbon glycerol backbone. This triglyceride form of the omega-3 fatty acids is the same form that is naturally used by the body to transport the omega-3 fatty acids in the blood circulation.
Note that the word “triglycerides” is commonly used in the field to both describe a particular form of omega-3 fatty acid composition, and also to describe blood fats in general, of which a positive effect of the omega-3 fatty acids is to lower the level of (non-omega-3) triglycerides that are circulating in the blood. To avoid confusion, “blood triglycerides” will be used to refer to the medical effect of the omega-3 fatty acids.
Although omega-3 fatty acids are predominantly found in unprocessed fish in a triglyceride form, natural, unprocessed fish oil is not generally used directly as a nutritional supplement due to safety concerns regarding unwanted environmental contaminants such as mercury. To avoid these unwanted contaminants, fish oil processors generally employ molecular distillation techniques to purify and/or concentrate the fish oil.
As a side effect of this molecular distillation process, most of the triglyceride form of the omega-3 fatty acids are converted to a synthetic (not-found in nature) ethyl ester form for encapsulation or bottling. That is, in the purification and concentration process, the various omega-3 fatty acid residue(s) are severed from the glycerol backbone of the triglyceride, and as a result of the process generally used, an artificial ethyl ester form of the omega-3 fatty acid is generated.
In the ethyl ester form, the terminal —OH group of the omega-3 fatty acid head carboxyl group is replaced by an ethyl alcohol —OH—CH2-CH3 group. This modification is considered harmless because once ingested; the user's liver can then, at least eventually, subsequently remove the synthetic alcohol group from the omega-3 fatty acid ethyl ester. However this conversion is neither 100% efficient nor instantaneous.
Alternatively, the manufacturer can, at a higher expense, take the ethyl-ester form of the omega-3 fatty acids, as well as glycerol and other fatty acids, and recreate an artificial omega-3 fatty acid in a triglyceride form that, to all intents and purposes is equivalent to the original natural omega-3 fatty acid in triglyceride form. Nordic Naturals, of Watson California, for example, produces their “Ultimate Omega” line of fish oil products using this approach. Due to the higher expense of this extra process, however, this approach is less common.
Because the ethyl ester form of the omega-3 fatty acids is cheaper to produce, and because manufacturers are understandably reluctant to publicize that they are providing an unnatural form of omega-3 in their nutritional supplements, unless fish oil label clearly says otherwise, it should be assumed that the fish oil contains a high proportion of the omega-3 fatty acids in the ethyl ester form.
Natural fish oil supplements thus generally consist of either the triglyceride form of EPA and DHA form of omega-3 fatty acids, or the synthetic ethyl ester form of EPA and DHA omega-3 fatty acids.
In addition to the natural triglyceride form of omega-3 fatty acids, the natural free fatty acid form of omega-3 fatty acids, and the artificial ethyl ester form of omega-3 fatty acids, another form of omega-3 fatty acids also exists, the phospholipid form.
The phospholipid form of omega-3 fatty acids is found in high concentrations in certain marine animal species adapted for life in extremely cold water, such as Antarctic krill (e.g. Euphausia superba). In this form, at least one omega-3 fatty acid residue, often in conjunction with a non-omega-3 fatty acid residue, is again attached to a glycerol backbone, but the third position on the glycerol backbone is occupied by a phosphate group, which in turn is usually conjugated with a choline, serine, or ethanolamine group. Such phospholipid forms of the omega-3 fatty acids likely help the krill continue to function at extremely low temperatures because they help keep the krill cell membranes fluid. In this respect, the phospholipid forms of the omega-3 fatty acids can somewhat considered to be acting like a natural version of a cell membrane antifreeze.
Thus in contrast to standard fish oil, the omega-3 fatty acids in purified Antarctic krill oil supplements generally consists of a mix of around 40%-50% phospholipids (which has omega-3 EPA and DHA carbon chains), and the rest of the omega-3 fatty acid groups are generally found either in the triglyceride form (natural form before molecular distillation) or in the ethyl ester form (after molecular distillation).
Although fish oil has long been a favorite of the nutritional supplement industry in various over-the-counter (non-prescription) forms, recently, the therapeutic utility of the omega-3 fatty acids has also attracted the attention of various pharmaceutical companies. For example, Lovaza, produced by GalaxoSmithKline, is a purified ethyl ester form of the omega-3 fatty acids EPA and DHA. Similarly Epanova, produced by Omthera Pharmaceuticals, is a purified free fatty acid form of the omega-3 fatty acids.
PPAR Receptors:
The omega-3 fatty acids are believed to mediate some of their actions, at least in part, by way of the peroxisome proliferator-activated receptors (PPARs). PPARs are nuclear receptor proteins that bind to retinoid hormones (e.g. hormones built around a carbon chain backbone that is somewhat similar to the carbon chain backbone of omega-3 fatty acids, such as prostaglandins, vitamin D, and relevant to this discussion, also omega-3 fatty acids in the non-esterified, free fatty acid form).
The PPAR receptor proteins are a family of proteins that exist in various forms, called the a, b, d, g1, g2, g3 form. After a PPAR receptor binds its particular ligand, it then forms a dimer with a retinoid Z receptor, and this complex in turn binds to the DNA of various genes, thereby regulating the transcription of these genes.
The PPAR a (alpha) receptors, for example, are primarily expressed in the liver and fat cells (adipose tissues), and play a critical role in both fat metabolism and diabetes. The PPAR g (gamma) receptors are also expressed in adipose tissues as well. Various drugs involved in regulating both triglyceride production and diabetes target various members of the PPAR family. For example, fibrate blood triglyceride lowering drugs target PPARa receptors. By contrast, various anti-diabetic drugs, such as the various thiazolidinediones (exemplified by drugs such as Avandia and Actos) target the PPARg receptors.
Mutations in PPAR receptors have been linked to lipid disorders, insulin resistance, and obesity.
As previously discussed, prior studies have suggested that the omega-3 or n-3 fatty acids may themselves interact with some PPAR receptors. For example, Jump, in “Dietary polyunsaturated fatty acids and regulation of gene transcription”, Curr. Opin. Lipidol. 2002 April; 13(2):155-64, reviewed various studies showing that non-esterfied (e.g. free, unbound to glycerol) fatty acids or fatty acid metabolites, in particular 18 and 20 carbon long fatty acids which are n-3 polyunsaturated fatty acids, may activate PPAR receptors, in particular PPARa.
PPARa (PPAR-alpha) is a transcription factor and a major regulator of lipid metabolism in the liver and other organs. Activation of PPARa promotes uptake, utilization, and catabolism of fatty acids by up-regulation and expression of genes involved in fatty acid transport and peroxisomal and mitochondrial fatty acid β-oxidation. PPARa is primarily activated through ligand binding such as by the free fatty acid form of omega-3 fatty acids.
It should be noted that when PPARa is stimulated, it changes the cell's genetic expression and metabolism to an alternate state which better enables fats to be burned as fuel. On the other hand, PPARg (PPAR-gamma) stimulation, as by current diabetic pharmaceuticals, results in the inhibition of fat burning, increased fat storage, and the manufacture of new fat cells. This effect of PPARg is obviously less than desirable for the majority of diabetics and the many overweight people throughout the world. Effective stimulation of PPARa for instance could, on the other hand, present powerful and beneficial benefits.
Caloric Restriction:
Another technique of regulating many metabolic activities in a favorable direction is caloric restriction. Caloric restriction has widely been recognized for over 70 years as being an effective means of prolonging maximal lifespan in many species, including mammals such as rodents, and even primates. As a result, some human enthusiasts have embraced this as a form of life extension protocol. However this life extension protocol, although possibly effective, is very hard to follow.
Caloric restriction diets, which are not generally considered appropriate for individuals under the age of 21, generally require human practitioners to eat between 10-25% fewer calories than average. These diets have been shown to produce impressive health results thus far in humans, including a reduction in cardiovascular disease markers as indicated by LDL particle number and size, coronary artery imaging techniques, carotid artery elasticity. Such diets also increase HDL, lower blood pressure, lower triglyceride levels, and are also associated with improved memory, and reduced inflammation. Because such diets are hard to follow on a long-term basis, however, there is great interest in finding methods to biochemically reproduce the desirable effects of caloric restriction without the hardships of caloric restriction.
Interestingly, recent work suggests that there may be a relationship between caloric restriction and the PPAR receptors. For example, Corton, et. al., “Mimetics of Caloric Restriction Include Agonists of Lipid-activated Nuclear Receptors”, J. Biol. Chem. 279 (44), 46204-46212 (2004) studied the impact of caloric restriction on normal and mutant PPARa-null mice. They found that the beneficial effects of caloric restriction were lacking in the PPARa-null mice, suggesting that the PPARa receptors may play a role in mediating the beneficial effects of caloric restriction.
Membrane Fluidity
On a side note, note, but relevant to this invention, a brief review of biological membranes is also in order.
Biological membranes are composed of a bilayer of membrane lipids, primarily cholesterol, glycolipids, and phospholipids. The lipid bilayer structure is thermodynamically favored because hydrophobic effects cause the lipid hydrocarbon chains to coalesce together to form an internal hydrophobic environment inside the membrane, while at the same time, the hydrophilic phosphate polar head groups of phospholipids face the exterior aqueous environment, thus creating a two dimensional lipid bilayer membrane structure.
Because the various lipid molecules are only weakly held into position in the membrane by hydrogen bonds, they are to some extent, free to move around within the two dimensional plane of the membrane. Thus biological membranes act in some respect like a two dimensional fluid. This fluidity is in fact an essential part of the proper biological operation of cells and cell membranes, because it allows embedded cell membrane proteins to move about in a two dimensional space and perform various functions that would otherwise not be possible if they were forced to be stationary.
The fluidity of a biological membrane varies, to some extent, depending on both ambient temperature and lipid composition. At cooler temperatures, such as those experienced by cold blooded marine animals, phytoplankton, and also plants, membrane fluidity is generally much less and more difficult to maintain than it would be in a warm blooded mammal environment.
Thermodynamically, what happens is that at lower temperatures, the hydrogen bonding between the different lipid molecules starts to dominate over the thermodynamic fluctuations that would otherwise cause these bonds to break. To cope with this problem, cold environment plants and marine animals incorporate a larger number of unsaturated omega-3 fatty acid residues in their membrane lipids. The double bonds in the omega-3 fatty acids tend to disrupt or not participate in the hydrogen bonding between different lipids, and thus promote membrane fluidity and again somewhat act like “anti-freeze” in this regard.
Absent specific transport mechanisms, such as transport proteins, pores, and the like, the cell membrane is generally fairly impermeable to most molecules, including free fatty acids, hormones, and the like. However workers, such as Lande et. al. Journal of General Physiology 106, 67-84 (1995) al., have noted that at least for some classes of molecules, higher membrane fluidity is positively correlated with increased permeability. Generally increased fluidity is also likely to help specific transport proteins and membrane receptors function with higher efficiency such as the glucose transporter complex GLUT4 and the insulin receptor, improving control of blood glucose and diabetes.